In-depth proteome characterization of endometrium and extraembryonic membranes during implantation in pig

Background Proteome characterization of the porcine endometrium and extraembryonic membranes is important to understand mother-embryo cross-communication. In this study, the proteome of the endometrium and chorioallantoic membrane was characterized in pregnant sows (PS) during early gestation (d 18 and 24 of gestation) and in the endometrium of non-pregnant sows (NPS) during the same days using LC-MS/MS analysis. The UniProtKB database and ClueGO were used to obtain functional Gene Ontology annotations and biological and functional networks, respectively. Results Our analysis yielded 3,254 and 3,457 proteins identified in the endometrium of PS and NPS, respectively; of these, 1,753 being common while 1,501 and 1,704 were exclusive to PS and NPS, respectively. In addition, we identified 3,968 proteins in the extraembryonic membranes of PS. Further analyses of function revealed some proteins had relevance for the immune system process and biological adhesion in endometrium while the embryonic chorion displayed abundance of proteins related to cell adhesion and cytoskeletal organization, suggesting they dominated the moment of endometrial remodeling, implantation and adhesion of the lining epithelia. Data are available via ProteomeXchange with identifier PXD042565. Conclusion This is the first in-depth proteomic characterization of the endometrium and extraembryonic membranes during weeks 3 to 4 of gestation; data that contribute to the molecular understanding of the dynamic environment during this critical period, associated with the majority of pregnancy losses. Supplementary Information The online version contains supplementary material available at 10.1186/s40104-024-01002-x.


Background
The female reproductive tract provides a unique environment for a successful pregnancy.It is imperative that the non-pregnant uterus be transformed into a capable environment for the establishment and maintenance of pregnancy.To achieve this, intimate cross-communication between the endometrium and the embryo is necessary at an early stage of life [1], and this dialog can influence subsequent fetal developmental potential [2] and even post-natal performance.Initially, the trophoblast-derived estrogen is one of the most important embryonic signals to activate the maternal uterus for attachment [3,

Animals
All experiments were conducted in compliance with international guidelines (Directive 2010/63/EU) and were approved by the Bioethical Committee for Animal Experimentation at the University of Murcia, Spain (research code: 522/2019; 2019/03/21).For this study, Landrace × Large-White sows, parity 2 to 7 (aged 1.5-3.5 years) with weight around 260-300 kg, were randomly selected from a commercial farm (Agropor SA, Murcia, Spain) immediately after weaning.
Mature boars (aged 2-3 years) with a proven record of fertility were selected as semen donors from a commercial breeding boar station (AIM Iberica, Spain).All animals were provided with ad libitum access to water and fed with commercial diets that met their nutrient requirements.

Estrus detection
Estrus detection was conducted as described previously [15] by snout-to-snout contact between females and a mature boar and applying back-pressure twice daily, starting one day after weaning.Only sows showing signs of estrus on d 4 after weaning were used for the experiment.

Experimental design
To characterize the proteomic profile of porcine endometrium and extra-embryonic membranes during early pregnancy, sows were post-cervically inseminated at 6 and 24 h after the onset of estrus (d 0 = onset of estrus) with 40 mL doses containing 1.5 × 10 9 live (for pregnant sows; PS) or dead spermatozoa (for non-pregnant sows; NPS, controls).The sows were euthanised on d 18 and 24 after insemination and their uteri were opened lengthwise along the anti-mesometrial side.In PS, pregnancy was verified by recovering conceptuses from both uterine horns, and both endometrial and extra-embryonic membranes samples were carefully cut out and pooled from 3 different areas of attachment in d 18 of gestation (n = 4 sows) or 3 different implantation areas in d 24 of gestation (n = 4 sows).In NPS, morphological examination of the ovaries immediately after euthanasia confirmed the presence of active corpora lutea (between 16-23 per sow) and endometrial tissues were cut out and pooled from 3 different areas at random of the uterine horn on d 18 (n = 4 sows) or d 24 (n = 4 sows).All samples were collected by the same technician, immersion-frozen in liquid nitrogen and preserved frozen at −80 ºC until analysis.

Proteomics analysis
Proteome analyses were conducted at the Proteomics Unit of the University of Valencia, which is affiliated with ProteoRed, PRB2-ISCIII, located in Valencia, Spain.

Spectral library building
For generation of a spectral library from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE; Bio-Rad, Richmond, CA, USA), aliquots containing the same amount of each sample type were combined to form a single pool.Endometrial samples from PS and NPS were divided into two different libraries with extra-embryonic samples kept in a separate library.
Gel protein digestion Each library's respective career was divided into 8 pieces and then subjected to digestion using Sequencing Grade Trypsin (Promega Corporation, Madison, WI, USA) following the method described by Shevchenko et al. [16].After trypsin digestion, 10% trifluoroacetic acid (TFA; Fisher Scientific, Madrid, Spain) was added to stop the reaction, and the resulting supernatant was removed.The sliced library gels were then dehydrated using pure acetonitrile (ACN; Fisher Scientific, Madrid, Spain).The resulting peptide solutions were combined with their corresponding supernatants, and the resulting peptide mixtures were dried in a speed vacuum.Prior to LC-MS/MS analysis, the dried peptides were resuspended in a solution containing 2% ACN and 0.1% TFA.

LC-MS/MS analysis
For the analysis, 5 μL of each digested pool was loaded onto a trap column (NanoLC Column, 3 μm C18-CL, 75 μm × 15 mm; Eksigent Technologies, Dublin, CA, USA) and desalted with 0.1% TFA at a flow rate of 3 μL/min for 5 min.Next, the peptides were loaded onto an analytical column (LC Column, 3 μm C18-CL, 75 μm × 12 cm, Nikkyo Technologies, Tokyo, Japan) that had been equilibrated with a solution containing 5% ACN 0.1% formic acid (FA; Fisher Scientific, Madrid, Spain).Peptide elution was carried out using a linear gradient of 5% to 40% of solvent B (0.1% FA in ACN) over a period of 120 min, with a flow rate of 300 nL/min.Finally, the eluted peptides were analyzed using a mass spectrometer nanoESIqQTOF (5600 TripleTOF, AB SCIEX, Framingham, MA, USA).
The TripleTOF was set to information-dependent acquisition mode.This involved conducting a 250-ms time of flight (TOF) MS scan within the range of 350-1,250 m/z, followed by 150-ms product ion scans within the range of 350-1,500 m/z on the 25 most intense ions with charges ranging from 2 to 5. To ensure consistency, the rolling collision energy equations were applied to all ions with a charge of 2+.These equations were determined by the following parameters: |CE|= (slope) × (m/z) + (intercept), where for 2+ ions, the slope was 0.0625 and the intercept was −3.

Protein identification
The peak list was generated using the ProteinPilot search engine (version 5.0 SCIEX) directly from the 5600 TripleTof wiff files.The Paragon algorithm of proteinPilot [17] was employed for searching the UniProt database with specific parameters, which included trypsin specificity, cys-alkylation, taxonomy mammalian restricted, and thorough search effort.FDR correction was also applied to ensure accurate protein identification.To prevent the use of identical spectral evidence in multiple proteins, the Pro Group algorithm was used to group the identified proteins.The Pro Group Report defined a protein group as a collection of proteins that share some physical evidence.Pro Group differs from sequence alignment analyses in that it relies solely on observed peptides for the formation of protein groups, rather than comparing complete theoretical sequences.As the observed peptides are derived from experimentally obtained spectra, the formation of protein groups in Pro Group can be viewed as being guided by the utilization of spectra.Consequently, unobserved regions of the protein sequence are irrelevant in elucidating the data.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [18] partner repository with the dataset identifier PXD042565.

Data analysis
The UniProtKB database (www.unipr ot.org) was used to conduct bioinformatic analysis of the identified endometrium and extraembryonic proteins.This analysis was aimed to acquire functional Gene Ontology (GO) annotation, specifically in the categories of "molecular function", "cellular component", and "biological processes".
Venn diagrams of the endometrial proteins were created to identify common and unique proteins of PS and NPS using Venny online software (https:// csbg.cnb.csic.es/ Bioin foGP/ venny.html).
Cytoscape 3.9.1 software and ClueGO v2.5.9 were utilized to generate an interaction network of unique proteins involved in immune system process, biological adhesion and reproductive process.The following selection criteria were applied: (1) a P-value cut-off of at least 0.05, (2) use of the enrichment/depletion statistical test (two-sided hypergeometric test) with Bonferroni step down, minimum GO level 2, maximum GO level 6, and a kappa score threshold of 0.4, and (3) analysis of Homo sapiens organism with genes present in KEGG pathways, REACTOME pathways and GO Biological Process.

Western blotting
In order to validate the LC-MS/MS results, total protein was extracted from the three pools of samples (endometrial samples from PS and NPS and extraembryonic samples) to perform Western blot analysis.Briefly, 100 mg of frozen tissues were disrupted in a polytron homogenizer using RIPA lysis buffer with protease and phosphatase inhibitor cocktails and quantified with the Bradford assay using BSA as standard (Protein Assay Kit, Bio-Rad, Hercules, CA, USA).Total protein extracts (30 mg) were mixed with 5 × SDS sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% b-mercaptoethanol, and 0.005% bromophenol blue) and resolved by SDS-PAGE on 10% acrylamide gels.Proteins were detected immunologically following electrotransfer onto 0.45-µm pore-size nitrocellulose membrane (Bio-Rad).The membranes were blocked with 5% BSA in TBS and 0.1% Tween-20 for 1 h at room temperature and incubated for 1 h with continuous agitation with primary antibodies.The antibodies used in this study were: antiheme oxygenase 1 (rabbit polyclonal, 1:2,000 dilution, Abcam, Cambridge, UK), anti-RPS6 (rabbit polyclonal, 1:5,000 dilution, Abcam), anti-PTN (goat polyclonal, 1:2,000 dilution, Abcam), and anti-thrombospondin-1 (rabbit polyclonal, 1:5,000 dilution, Abcam).Blots were washed three times for 10 min in TBS and 0.1% Tween-20 and incubated with horseradish peroxidase-conjugated (HRP) secondary antibodies for 1 h at room temperature with continuous agitation: HPR-conjugated goat anti-rabbit IgG antibody (1:5,000 dilution, Merck KGaA, Darmstadt, Germany) and HPR-conjugated rabbit antigoat IgG antibody (1:5,000 dilution, Merck KGaA).Blots were developed using a peroxidase reaction with the enhanced chemiluminescent immunoblotting detection system (ECL-Plus, GE Healthcare, Little-Chalfont, Buckinghamshire, UK).Antibodies were accepted when they displayed a single predominant band at the expected molecular weight.

Analysis of the endometrial proteome profile
Using FDR correction, the UniProt mammalian library enabled the identification of 3,254 proteins in the endometrium of PS during the peri-implantation period (d 18 and 24 of gestation).Similarly, 3,457 proteins were identified in the endometrium of NPS on the same days of the cycle.Additional file 1: Tables S1 and S2 contain a comprehensive list of all proteins identified, along with their corresponding unused score, percentage of sequence coverage, UniProt accession number, protein name, species, and matched peptides.Both endometrial proteomes featured high-abundance proteins (Tables 1 and 2) that were primarily associated with ion transport and binding.Examples include albumin, hemoglobin subunit beta, and globin domain-containing protein.Both endometrial proteomes also contained proteins related to cytoskeleton organization, such as myosin-9 and plectin, while filamin A was exclusive to the endometrial proteome of PS.Proteins involved in inflammatory and immune responses of the endometrium, such as complement C3 and IgG heavy chain proteins, were also present in both PS and NPS proteomes, while the proteome of NPS also included Ig-like domain-containing protein.

Gene Ontology analysis of the endometrial proteome
Over the 3,254 proteins identified in the UniProt mammalian endometrial library of PS, a total of 3,122 proteins were successfully mapped to UniProtKB IDs, from which 2,484 proteins had biological process annotation information (Fig. 1A).The highest proportion included cellular and metabolic processes and biological regulation.However, there were also 349, 268, 115 and 62 proteins involved in developmental process, immune system process, biological adhesion, and reproductive process, respectively.Most of the proteins with reproductive process function were related to developmental processes associated with reproduction and multi-organism and multi-cellular organismal reproductive processes (Fig. 1B).There were also 16 and 12 proteins enclosed to fertilization and cellular process involved in reproduction, respectively and two proteins, alpha-2-macroglobulin and platelet-derived growth factor receptor alpha related to luteinization term.When the proteins with information on molecular function were analyzed, most of them were enclosed into binding and catalytic activity (2,408 proteins; Fig. 1C).Out of the 2,474 proteins with cellular components information, most of them were associated with the cellular anatomical entity (Fig. 1D).
A similar GO functional classification was evident in the endometrium of NPS.Of the 3,457 proteins identified in the UniProt mammalian library, a total of 3,348 proteins were successfully mapped to UniProtKB IDs.Among these, 2,658, 2,547 and 2,685 proteins were annotated with information related to biological process, molecular function and cellular component, respectively (Fig. 2).Out of the 68 proteins that were found to contain information regarding reproductive process in the NPS (Fig. 2B), 10 proteins had meiotic cell cycle annotation, while 3 proteins were related to the ovulation cycle process (myosin-9, progesterone receptor and plateletderived growth factor receptor alpha).
We compared the two endometrial proteomes to check identified proteins that were unique to either the PS or the NPS proteome, as well as those that were common to both.As a result, we found that 1,753 proteins were detected in both groups of samples, while 1,501 proteins were exclusively detected in PS samples, and 1,704 proteins were solely identified in NPS samples.The lists of proteins identified in both groups and found only in PS and NPS samples are available in Additional file 1 (Tables S3-S5).In this context, and to clarify the proteomic profile of both groups, we perform the categorization of the unique proteins according to the protein class ontology.Upon conducting a functional GO analysis of the unique proteins present in the PS endometrial samples using the UniProtKB database, it was observed that 53 and 23 proteins were involved in immune system process and biological adhesion, respectively (Tables 3 and 4).In addition, there were 17 PS-unique proteins such as MAPK14 (mitogen-activated protein kinase 14), MAP2K1 (mitogen-activated protein kinase kinase 1), HECTD1 (HECT-type E3 ubiquitin transferase), HSD17B2 (estradiol 17-betadehydrogenase 2), HPGD (15-hydroxyprostaglandin dehydrogenase), RPS6 (40S ribosomal protein S6) and DLG1 (discs large MAGUK scaffold protein 1), which were found to play important roles in reproductive processes, specifically in relation to the establishment and maintenance of pregnancy (Table 5).The functional analysis of the unique proteins that were identified in the NPS endometrium samples, revealed that 58, 25, and 25 proteins were involved in immune system process, biological adhesion and reproductive process, respectively.It was observed that some of these proteins played important roles in the development of the reproductive cycle (Tables 6, 7 and 8).

Analysis of the extraembryonic membrane proteome profile
The UniProt mammalian library with FDR correction allowed the identification of 3,968 proteins in extraembryonic membranes of PS during the peri-implantation period (d 18 and 24 of gestation).The list of all identified proteins, including their unused score, percentage of sequence coverage, UniProt accession number, protein name, species and matched peptides can be found in Table S13.Table 9 shows the ten most highly abundant proteins identified in the extraembryonic membranes.This group included some critical extracellular matrix proteins, such as fibronectin, which is a multifunctional adhesive glycoprotein with a relevant role in embryogenesis, and heparan sulfate proteoglycan 2, which interacts with many other proteins and has many functions in cell signaling, adhesion and angiogenesis.Other proteins  Of the proteins identified in the extraembryonic membranes, a total of 3,808 proteins were successfully mapped to UniProtKB IDs, with 3,114, 3,014 and 3,109 proteins annotated with biological process, molecular function and cellular component annotation, respectively (Fig. 7).Within the proteins that were annotated with biological process, 132 proteins were involved with biological adhesion annotation (Table 10) and 94 proteins were associated with reproductive process (Fig. 7B, Table 11).

Validation study
To validate the results of our qualitative LC-MS/MS proteomic study, 4 of the identified proteins were selected for Western blot analysis (Fig. 10).The selected proteins were HMOX1 (heme oxygenase 1) and RPS6 (40S ribosomal protein) proteins from the endometrium of PS, PTN (pleiotrophin) protein from the endometrium of NPS and THBS1 (thrombospondin 1) protein from the extraembryonic membranes.This selection was based on the availability of the antibodies for the study of porcine proteins and on the important role of HMOX1 and RPS6 in the development of endometrial receptivity in the case of PS proteins.The Western blot validation experiment confirmed the presence of HMOX1 and RPS6 proteins in the endometrium of PS and PTN protein in the endometrium of NPS as well as the presence of THBS1 protein in the extraembryonic membranes, supporting the proteomic data.

Discussion
This study presents a comprehensive characterization of the endometrial proteome during d 18 and 24 of both PS and NPS, with the major objective of creating a catalog of proteins that are present during this crucial phase of porcine gestation.This period is important as it occurs immediately after trophoblast attachment and marks evidence of early pregnancy.This study is the first of its kind to perform such a large-scale characterization.We also analyzed the proteomic profile of extraembryonic membranes to better understand the functionality of the placenta at this critical stage of pregnancy.Proteomics is a highly dynamic element that changes in an organism, tissue, or cell because of environmental changes, stress, or a physiological or pathological condition.It provides valuable information that cannot be Fig. 3 Visualization of the reactome pathways analysis of unique proteins involved in immune system process (A) and biological adhesion annotation (B) in pregnant sows.The size of the nodes indicates the pathway´s enrichment significance.The following ClueGO parameters were used: P-value cut-off = 0.05; Statistical test used the enrichment/depletion (two-sided hypergeometric test); Bonferroni step down with minimum GO level 2; maximum GO level 6; kappa score threshold equal to 0.4.GO = Gene Ontology.Network (A) includes the interaction of CCR5, HMOX1, IFI35, ISG15, LBP, MAP2K1, MAPK14, SLA, STXBP2, VAMP7, CD14, DNM1, LBP, MAP2K1, MAPK14 and S100A9 proteins derived from genomic or transcriptomic studies [19].Quantitative analysis of proteins is essential to obtain relevant information on specific biological system; however, to obtain an overall view of the proteomic profile, qualitative proteomic analysis is also required.This qualitative information could contribute to a better understanding of the local maternal response during implantation and provide possible clues to reduce pregnancy failure.For our proteomic study, we used LC-MS/MS operated in datadependent acquisition mode and it supported the detection of 3,254, 3,457 and 3,968 proteins at high-confidence in PS and NPS endometria and membranes, respectively.
Previous reports have identified endometrial proteins during various pregnancy time points using different proteomic techniques.The most classically used has been the two-dimensional (2D) gel electrophoresis followed by mass spectrometry (MS) or MALDI TOF-TOF analysis to identify protein spots.The 2D-MS/MALDI TOF-TOF has been used to identify 70 to 500 proteins in the peri-implantation period (d 9 to 16) and 820 proteins at mid-pregnancy (d 40 to 93) in the endometrium of pregnant and non-pregnant sows [11-13, 20, 21].These proteomic studies have provided information on the dynamic physiological protein profile and how it may relate to maternal recognition and pregnancy.In recent years, advances in proteomics, particularly technologies based on MS, have allowed us to increase sensitivity, confidence and proteomic coverage compared to those of traditional methods.Using LC-MS/MS, we were able to identify a substantial number of proteins in our study.These findings are consistent with the 3,185 proteins that were previously identified by Wang et al. [14] in endometrial tissue during mid-pregnancy.
When we explored the endometrium proteome of PS and NPS, we first observed that the 10 high abundant proteins found in the two proteomes were proteins involved in immune system process and proteins with binding and cytoskeletal organization functions, both of which are involved in the structural support of the endometrium.In general, the functions of these 10 proteins are consistent with the basic development of the endometrium, which is constantly remodeling.To further classify the identified proteins in the endometrial tissues, we conducted an evaluation of their functional gene ontology in each endometrial proteome.As expected, both proteomes exhibited a similar pattern of functional categorization.For this reason, and to identify exclusive proteins during these critical days of pregnancy, we created a Venn diagram by comparing all proteins identified in the endometrium of PS with all proteins identified in the NPS endometrium during the corresponding days of the cycle.To the best of our knowledge, this study provides the largest number of unique proteins identified in the endometrium of PS and NPS during the peri-implantation period (1,501 and 1,704 proteins, respectively).The proteins identified in these two lists may contribute to a better understanding of the biology of the endometrium during the implantation phase and serve as valuable tools for identifying the main regulators responsible for endometrial remodeling and the appropriate development of pregnancy.To gain a more extensive insight into the biological networks of these distinctive proteins, we utilized ClueGO, which is a Cytoscape plugin that greatly enhances the biological interpretation of genes and proteins by selecting relevant GO terms Fig. 5 Visualization of the reactome pathways analysis of unique proteins involved in immune system process (A) and in biological adhesion annotation (B) in non-pregnant sows.The size of the nodes indicates the pathway´s enrichment significance.The following ClueGO parameters were used: P-value cut-off = 0.05; Statistical test used the enrichment/depletion (two-sided hypergeometric test); Bonferroni step down with minimum GO level 2; maximum GO level 6; kappa score threshold equal to 0.4.GO = Gene Ontology.Network (A) includes the interaction of ABI1, C5AR1, CD44, CFD, CFP, ELANE, HP, HRAS, IFIH1, MMP9, MYD88, PSMB6 and VNN1 proteins.Network (B) includes the interaction of FN1, ITGA3, ITGA9, ITGB4, LAMA3, LAMA5, THBS4, TNXB, EGFR, ITGAX, EGFR, PARVA, and TNXB proteins and pathways and displaying them in well-organized networks.
The unique proteins involved in the immune system of PS are of particular interest, given that the modulation of the endometrial immune system is crucial for endometrial function and implantation [9,22].Among the 53 PS-unique proteins annotated, 10 proteins were involved in cytokine signaling in immune system pathway in ClueGo analysis.The immunological status of the female reproductive tract is dynamic and subject to changes depending on both the estrous cycle and gestational stage.Research has shown that the mid-implantation  6 Visualization of the biological gene ontology analysis of unique proteins involved in the reproductive process annotation in non-pregnant sows.The size of the nodes indicates the pathway's enrichment significance.The following ClueGO parameters were used: P-value cut-off = 0.001; Statistical test used the enrichment/depletion (two-sided hypergeometric test); Bonferroni step down with minimum GO level 2; maximum GO level 6; kappa score threshold equal to 0.4.GO = Gene Ontology stage (d 18) is characterized by an increase in pro-inflammatory cytokine expression, which facilitates embryo implantation interactions between the placenta and uterus during porcine pregnancy [23,24].Conversely, during the late implantation phase (d 20-28), antiinflammatory expression is observed to support placental and conceptus development [22].Practically all identified PS-unique proteins involved in immune system process were pro-inflammatory proteins, with the exception of HMOX1, which has anti-inflammatory properties.Based on this, it is possible that a pro-inflammatory environment predominates from d 18 to d 24 of gestation, with a rapid shift towards an anti-inflammatory environment by d 25.Furthermore, we also identified that several PS-unique proteins of PS endometrium were involved in the Toll Like Receptor 4 (TLR4) cascade pathway.Toll-like receptors are important sensors of the innate immune system that promote the production of important pro-inflammatory molecules such as cytokines and chemokines [25].Activation of TLR4 during pregnancy has been shown to bridge innate and adaptive immunity to protect the developing fetus from pathogens [26].Among the identified PS-unique proteins involved in the TLR4 cascade, LBP and CD14, are pattern recognition receptors or proteins capable of recognizing molecules commonly found in pathogens and acting as the first line of defense [27].Additionally, MAPK14 and MAP2K1 proteins, play an important role in the cascades of cellular responses that lead to the direct activation of transcription factors implicated in inflammatory responses [28].A healthy pregnancy requires a balance between immunological defense and immunosuppression to ensure semi allogenic fetal development.The involvement of the PS endometrial proteins identified in our study in the TLR4 cascade may indicate that an active maternal immune system is crucial to combat the upcoming intrauterine microorganism challenge.Herein, we have identified several proteins that may contribute to the modulation of the endometrial immune system, at least between d 18 and 24 of gestation.
The implantation and maintenance of pregnancy is closely related to the restructuring of the cytoskeleton and the remodeling of adhesive contacts with the extracellular matrix (ECM) [29].Based on their biological adhesion annotation, all PS-and NPS-unique proteins of the endometrium are proteins with different structural functions that control cell behavior such as proliferation, adhesion, and migration and regulate cell differentiation and death.The analysis by Cytoscape ClueGO of the NPS-unique proteins showed that most of the proteins with biological adhesion annotation were involved in focal adhesion, ECM-receptor interaction, PI3K-Akt signaling and regulation of actin cytoskeleton pathways.The laminin (LAMA3, LAMA5) and integrin (ITGA9, ITGAX, ITGB4, ITGA3) proteins, as well as FN1 and other NPS-unique proteins involved in these pathways, are implicated in cyclic remodeling of the endometrial epithelium in the absence of embryo implantation.
Regarding the PS-unique proteins identified in the biological adhesion category of the present study, these proteins are associated not only with the organization of the cytoskeleton but also with the adhesion process.
Of the 23 PS-unique endometrial proteins, Cytoscape ClueGO analysis revealed that EMILIN1, HAPLN1, TNC, and ITGAM are involved in the network of ECM organization.EMILIN1 protein plays a role in creating elastic fibers and promoting cell adhesion.This protein has been identified as a ligand molecule in the endometrium that interacts with receptors found on bovine d 16 conceptuses [30].The protein TNC is involved in embryogenesis and plays an important role in endometrial proliferation [31].It has been established that the integrin gene ITGAM also plays a crucial role during implantation, as the injection of an ITGAM antibody into the uterine lumen of early pregnant mice results in pregnancy loss [32].The expression of this gene, which is related to cell adhesion processes, was found to be upregulated in the luminal epithelium of the pig endometria collected at d 14 of pregnancy [33].HAPLN1 is another well-known ECM protein that plays a role in the adhesion process and is also expressed in various tissues such as the intestine and human placenta [34].In addition to the above proteins, in the present study we identified other unique proteins in PS endometrium that may be important for the adhesion of embryos to the surface of the endometrium.One of these proteins is the TGFB1.Among other processes, TGFB1 has an important function in angiogenesis, embryogenesis and trophoblast attachment [35] and its activation at d 15-16 of pregnancy in pigs was suggested by Kolakowska et al. [20].We confirm the presence of TGFB1 in the endometrium of PS at d 18-24 of gestation, which may indicate its importance in the proper course of pregnancy.In addition, the detection of high expression levels of HAPLN1 and TGFB1 in the endometrium of pregnant sows at mid-pregnancy [14] indicates that these proteins could play a crucial role in both implantation and the maintenance of pregnancy.The PS endometrial proteins with biological adhesion annotation identified in the present study, could be candidate proteins potentially involved in embryo-maternal dialogue.
The proteins with reproductive process annotation are particularly important for the interpretation of the PS proteome.Among these, we have identified proteins that are involved in and contribute to the successful establishment of pregnancy.Proteins such as RPS6, HECTD1, HSD172B2, MAP2K1 and MAPK14 are involved in ClueGO analysis in the reproductive structure and system development, and placenta development GOs, and could be responsible for the development of endometrial receptivity.It has been reported that RPS6 is a downstream target protein of the phosphatidylinositol-3-kinasa-AKT (PI3K/AKT) pathway.This signaling system plays an important function during the implantation phase of porcine pregnancy by stimulating the migration and attachment of trophectoderm and luminal epithelial  cells [36].HECTD1, identified here in PS endometrial tissue, has been previously described as indispensable for normal embryogenesis and fetal survival in mice [37] and as an important protein for human [38] and mouse [39] placentation.Therefore, it is reasonable to assume that this protein may be involved in the development of the junctional zone between the endometrium and extraembryonic membranes in pigs.High expression of HSD17B2 has been observed in the horse endometrium [40].This molecule exerts a local antiestrogenic effect by converting active estradiol to inactive estrone in endometrial epithelial cells [41].MAPK14, which is involved in mitogen-activated protein kinase pathways, mediates embryonic responses and plays an important role in the growth of human embryos [42].Similarly, MAP2K1 is important for the full expansion of the fetal-maternal exchange area and its loss of function leads to embryonic death due to placental defects [43].In addition, we have identified other PS-unique proteins involved in the development of pregnancy, such as HPGD and DLG1.It has been reported that the expression and activity of HPGD is regulated by the progesterone and that this molecule may be involved in the protection of pregnancy from prostaglandin at the beginning of pregnancy [44].The involvement of DLG1 protein in pregnancy events is completely unknown, but it has been shown to be involved in dynamic tissue movement [45] and its loss of function leads to complete neonatal lethality [46].In the pig, as in other mammals, the establishment of reciprocal interactions between the embryo-fetus and its associated extraembryonic membranes, particularly the chorio(allantois) and the endometrium is necessary for the success of implantation and the maintenance of a healthy pregnancy.The placenta in the pig is not Fig. 8 Visualization of the functional analysis of the proteins involved in the reproductive process annotation in extraembryonic membranes of pregnant sows at d 18 and 24 of gestation.The size of the nodes indicates the pathway´s enrichment significance.The following ClueGO parameters were used: P-value cut-off = 0.001; Statistical test used the enrichment/depletion (two-sided hypergeometric test); Bonferroni step down with minimum GO level 2; maximum GO level 6; kappa score threshold equal to 0.4.GO = Gene Ontology.Network includes the interaction of RAD21, SMC1A, SMC3, CCT3, CCT4, CCT8 and TCP1 proteins invasive.As a type of epitheliochorial placenta, around d 12-13 of gestation, the adhesion of embryos to the uterine epithelium starts [47,48].In the present study, we identified for the first time the proteome of extraembryonic membranes of PS at early stage of pregnancy (d 18 and 24).This study is primary descriptive and these proteins can be a good starting point for the discovery of proteins related to placental development and the implantation process.As expected, the functions of the high abundance proteins in the extraembryonic membranes were related to trophoblastic cell proliferation and placental tissue organization [49], as well as vascularization and angiogenesis, which are necessary for utero-placental blood flow to supply nutrients to the fetus [50].Out of the 3,968 proteins identified, we were interested in the 94 proteins annotated with reproductive processes.Our goal was to identify and characterize the protein-protein interaction networks that govern placental function.In this study we were particularly interested in the interaction among proteins related placenta and embryo development terms.Among them, HSD17B2, MAPK14, ITGB4, and DLG1 could be related to the balanced coordination of signaling pathways between membranes, endometrium, and embryo-fetus because they are also expressed in the endometrium of PS.The current network results could be important for identifying significant proteins as candidates for future functional studies of the placenta.Of potential interest is the identification of the protein NOTCH2.During early gestation, the vascular development of the fetus and extraembryonic tissues has been found to be significantly impacted by Notch signaling molecules.For instance, the smaller size of cloned porcine extraembryonic tissues at d 26 of gestation, which ultimately leads to embryonic losses in the first trimester, has been attributed to the low expression of these molecules [51].S2.Complete list of the 3,457 proteins identified in endometrium of non-pregnant sows at d 18 and 24 of cycle validated with a peptide confidence threshold of 95% and a false discovery rate (FDR) of 1%.Table S3.Complete list of the 3,968 proteins identified in extra-embryonic membranes of pregnant sows at d 18 and 24 of gestation validated with a peptide confidence threshold of 95% and a false discovery rate (FDR) of 1%.Table S4.Complete list of 1,751 unique proteins in endometrium of pregnant sows.Table S5.Complete list of 1,704 unique proteins in endometrium of non-pregnant sows.Table S6.
Complete list of 1,753 common proteins in endometrium of pregnant and non-pregnant sows.Table S7.Functional group analysis with their associated proteins in unique pregnant sow's proteins with immune system annotation (data generated by ClueGO plugin).Table S8.Functional group analysis with their associated proteins in unique pregnant sow's proteins with biological adhesion annotation (data generated by ClueGO plugin).Table S9.Functional group analysis with their associated proteins in unique pregnant sow's proteins with reproductive process annotation (data generated by ClueGO plugin).Table S10.The Gene Ontology (GO) Biological functional groups analysis with their associated proteins in unique pregnant sow's proteins with reproductive process annotation (data generated by ClueGO plugin).Table S11.Functional group analysis with their associated proteins in unique non-pregnant sow's proteins with immune system annotation (data generated by ClueGO plugin).Table S12.Functional group analysis with their associated proteins in unique pregnant sow's proteins with biological adhesion annotation (data generated by ClueGO plugin).Table S13.The Gene Ontology (GO) Biological functional groups analysis with their associated proteins in unique pregnant sow's proteins with reproductive process annotation (data generated by ClueGO plugin).Table S14.Functional group analysis with their associated proteins in pregnant sow's extraembryonic membrane proteins with reproductive process annotation (data generated by ClueGO plugin).Table S15.The Gene Ontology (GO) Biological functional groups analysis with their associated proteins in pregnant sow's extraembryonic membrane proteins with reproductive process annotation (data generated by ClueGO plugin).

Fig. 1
Fig. 1 Gene Ontology analysis of the endometrial proteome of pregnant sows during implantation.A Biological process.B Reproductive process.C Molecular function.D Cellular component

Fig. 2
Fig. 2 Gene Ontology analysis of the endometrial proteome of non-pregnant sows at d 18 and 24 of the cycle.A Biological process.B Reproductive process.C Molecular function.D Cellular component

Fig. 4
Fig.4 Visualization of the biological gene ontology analysis of unique proteins involved in the reproductive process annotation in pregnant sows.The size of the nodes indicates the pathway´s enrichment significance.The following ClueGO parameters were used: P-value cut-off = 0.001; Statistical test used the enrichment/depletion (two-sided hypergeometric test); Bonferroni step down with minimum GO level 2; maximum GO level 6; kappa score threshold equal to 0.4.GO = Gene Ontology.Network include the involvement of HECTD1, HSD17B2, MAP2K1, MAPK14, RPS6, MSH2, DLG1 and INHBB proteins among others

R
Fig.6 Visualization of the biological gene ontology analysis of unique proteins involved in the reproductive process annotation in non-pregnant sows.The size of the nodes indicates the pathway's enrichment significance.The following ClueGO parameters were used: P-value cut-off = 0.001; Statistical test used the enrichment/depletion (two-sided hypergeometric test); Bonferroni step down with minimum GO level 2; maximum GO level 6; kappa score threshold equal to 0.4.GO = Gene Ontology

Fig. 7
Fig. 7 Gene Ontology analysis of the extraembryonic membranes proteome of pregnant sows at d 18 and 24 of gestation.A Biological process.B Reproductive process.C Molecular function.D Cellular component

Table 1
Summary of high abundance proteins in the endometrium of pregnant sows (PS) during implantation

Table 2
Summary of high abundance proteins in the endometrium of non-pregnant sows (NPS) at d 18 and 24 of the cycle

Table 3
Complete list of exclusive proteins involved in immune system process in the endometrium of pregnant sows (PS) during implantation

Table 4
Complete list of exclusive proteins involved in biological adhesion annotation in the endometrium of pregnant sows (PS) during implantation

Table 5
Complete list of exclusive proteins with reproductive process annotation in the endometrium of pregnant sows (PS) during implantation

Table 6
Complete list of exclusive proteins involved in immune system process in the endometrium of non-pregnant sows (NPS) at d 18 and 24 of cycle

Table 7
Complete list of exclusive proteins involved in biological adhesion annotation in the endometrium of non-pregnant sows (NPS) at d 18 and 24 of cycle

Table 8
Complete list of exclusive proteins with reproductive process annotation in the endometrium of non-pregnant sows (NPS) at d 18 and 24 of cycle

Table 9
Summary of high abundance proteins in extra-embryonic membranes of pregnant sows (PS) during implantation

Table 10
Complete list of extraembryonic membranes proteins of pregnant sows (PS) involved in biological adhesion annotation

Table 11
Complete list of extra-embryonic membranes proteins with reproductive process annotation